Porcine reproductive and respiratory syndrome virus (PRRSV) is a small, enveloped, positive-stranded RNA virus which belongs to the genus arterivirus. PRRSV was isolated in Europe and in the United States of America in 1991 (Collins et al., 1992, Wensvoort et al., 1991). The genus arterivirus also includes equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV) and simian hemorrhagic fever virus (SHFV). The genus arterivirus is the only member in the family of the Arteriviridae. The family Arteriviridae and the Coronaviridae are grouped into the order Nidovirales.
In vivo, PRRSV reproduces primarily in porcine alveolar lung macrophages and blood monocytes. (Wensvoort et al., 1991). Macrophages from other tissues, such as heart, tonsil, spleen, turbinates and choroid plexus are also susceptible to PRRSV infection.
In vitro, the porcine virus can be passaged to primary cultures of porcine alveolar lung macrophages and blood monocytes, to cultures of the African green monkey kidney cell line MA-104 and to the derivative cell lines CL2621 and MARC-145. (Collins et al., 1992, Wensvoort et al., 1991). A few cell lines exist that the porcine virus cannot penetrate, such as BHK-21 and Vero cells, but once the genome of these cell lines is brought inside the cells artificially, the porcine virus is able to replicate and produce new virus particles. (Meulenberg et al., 1998).
PRRSV enters porcine alveolar macrophages and MARC-145 cells via receptor-mediated endocytosis. The uptake of PRRSV by the host cell is a multi-step process in which one or two viral constituents appear to interact with at least one host proteonic factor before virus-containing clathrin-coated pits are formed. (Kreutz, 1998). The entry process of PRRSV into porcine macrophages differs from the binding and internalization of PRRSV into MARC-145 cells. A membrane protein of about 210-kDa has been identified as being a putative receptor for PRRSV on macrophages (Duan et al., 1998) while a heparin-like molecule is suggested to be important for the binding of PRRSV to MARC-145 cells. (Jusa et al., 1997).
PRRSV has a concise genome structure (for a review, see, Snijder, and Meulenberg, 1998) which is 15,098 nt in length without the poly-A tail and contains 9 open reading frames (ORFs) flanked by a 5′ and 3′ non-translated region (NTR) of 221 and 114 nucleotides, respectively. The two 5′-terminal ORFs designated 1A and 1B (FIGS. 1A and 1B) comprise about 75% of the genomic RNA. The structural region of the RNA contains 7 ORFs designated 2A, 2B, and 3 to 7 (FIGS. 1A and 1B) and occupies the 3′-terminal third of the genome. ORF 1A/B encodes the non-structural proteins (nsps) involved in replication of the RNA genome and the forming of a 3′ nested set of subgenomic RNAs (sgRNAs).
The first two N-terminal proteins named nsp 1α and nsp 1β, respectively, have been studied for PRRSV. The functions of the other nsps are based on predictions derived from sequence homologies with EAV. Nsp 1α and nsp 1β are papain-like cysteine proteases and together with nsp2 and nsp4, a cysteine protease and a serine protease, respectively, the entire polyprotein ORF1 (a/ab) is assumed to be cleaved into 12 nsps. Nsp9 and 10 are predicted to be the replicase subunits.
The 3′-terminal end of the genome encodes the structural proteins. The ORFs 2A to 6 encode six structural membrane-associated proteins and the most 3′-terminal ORF encodes the N protein (ORF7). The minor glycoproteins are GP2a (formerly named GP2), GP3 and GP4. The GP2a and the GP4 proteins are class I membrane proteins and have molecular masses of 27-30 and 31-35 kDa, respectively. Both N-glycosylated proteins are constituents of the virion. The other minor N-glycosylated structural protein of 42-50 kDa is GP3 and is encoded by ORF3. The nature of GP3 was unclear until the present disclosure.
The three major structural proteins are GP5, M and the N protein. GP5, a N-glycosylated protein of 24-26 kDa, is the most variable protein in its sequence. The most conserved protein is the non-glycosylated M protein. The M protein is a 18-19 kDa integral membrane protein and appears to have a function in viral infectivity. The M protein forms a heterodimer with GP5 which is present in the virion. The N protein is a small, highly basic, nucleocapsid protein of 14-15 kDa which interacts with the viral RNA during assembly. (Snijder & Meulenberg, 1998).
Sequence comparisons and studies involving the antigenic nature of PRRSV strains reveal two distinct groups designated as EU (European) and US (North American) strains. (Wensvoort et al., 1992, Snijder & Meulenberg, 1998). Another indication of the observed diversity between the EU strain and the US strain from the two continents is the fact that EU field isolates initially replicate easier in alveolar lung macrophages, whereas US isolates are recovered easier in the African green monkey kidney cell line MA-104 or derivatives thereof, such as CL2621 or MARC-145 cells. (Bautista et al., 1993).
The candidate proteins which will adsorb to the host cell surface of permissive cells are GP2a, GP2b, GP3, GP4, GP5, the M protein or a heterodimer of GP5 and the M protein.
Typically, when viruses are replicated in vitro in cell lines derived from a different species, the viruses tend to attenuate in the process of adapting to the cell line. This adaptation characteristic is widely used in vaccine development and is also true for PRRSV replicated in monkey cell lines (Collins et al. 1992).